All optical microscopes require a mechanism by which to provide focus in order to clearly image a specimen. In many instances, the system focus depends upon a specimen having visually recognizable features. These features produce necessary contrast for focus. In other instances, the distance to the specimen is determined and the focus set by the known focal length of the objective. Transparent specimens present unique challenges for establishing proper focus, and special techniques known only to highly skilled practitioners in the art are required to improve contrast to a point where proper focusing is possible.
Microscopes are traditionally focused by increasing or decreasing the distance between the microscope objective lens and the specimen until the image appears in focus to the microscopist. The focusing of a microscope is thus typically somewhat subjective, and microscopes must have focus-adjusting capabilities in order to properly visualize surface features of a specimen. Most often, a knob is provided to move either the objective lens or the object stage holding the specimen, in order to adjust the relative distance between the objective lens and the specimen, and the microscopist manipulates this knob until he subjectively believes that the best focus has been reached. Thus, despite the need for ever finer accuracy, the microscopist essentially remains the subjective tool by which focus is subjectively determined.
Technology has taken the subjectivity out the determination of focus by two basic methods. A first method measures the distance between the objective and the specimen by sensing light, such as near-infrared, reflected from the surface of the specimen to provide feedback to automatically control the focus. This type of method is described by Nikon Corporation (Japan). In another method, Nikon also describes the use of reflected sound to measure distance. Laser focus control systems as described by MotionX Corporation (U.S.), for example, in their FocusTrac Laser Auto Focus Systems, and Prior Scientific (U.S.), also facilitate focus by measuring the distance between the objective and the specimen and providing feedback for focus adjustment. U.S. Pat. No. 7,345,814 further describes a laser focus system. In some cases, these methods have difficulty focusing on transparent specimens due to the specimen reflectivity and transparency. These distance measuring methods generally require the addition of hardware and control software to facilitate focus using a standard microscope. U.S. Pat. No. 5,594,235 uses a confocal sensor to optimally measure the height, or Z axis point as it is referred to in the prior art. A given number of z points are pre-determined, and then each is evaluated for reflectance. Due to the varying degrees of reflectance, height at each point of the surface can be determined. While this may be thought of as a tool for topographical measurement, it also guarantees a certain confidence of focus accuracy.
A second known method to focus a microscope is to compare the contrast of an image as the distance between the microscope objective and specimen is increased and decreased. This is basically the method that a microscopist uses to visually determine that a specimen is in focus, as described above. The method is easily automated by capturing the specimen image electronically using a sensor such as, but not limited to, CCD or CMOS sensors. This method is described in detail by Groen and Young, et al, in “A Comparison of Different Focus Functions for Use in Autofocus Algorithms,” Cytometry 6:81-91 (1985). However, this method of contrast comparison cannot be used on specimens which are fully transparent or which do not have contrast within the image.
Methods using a microscope's field aperture, F-Stop, are employed to focus upon a specimen. This method is described by Chow and Liu, Nikon Optical Microscope, Basic Operation Procedure, http://nanomech.me.washington.edu/pdfs/nikon.pdf, January 2009, and further described in Nanometrics Incorporated's NanoSpec™ 3000 Lab Manual, NanoSpec Film Thickness Measurement System, Chapter 8.33. In this method, the F-Stop is closed in order to project the F-Stop on the specimen, and the focus is adjusted (i.e., specimen and objective are relatively moved), such that the contrast increases and/or decreases. At the point of greatest contrast, the specimen is considered in-focus and the F-Stop is opened to allow imaging of the specimen.
In microscopic imaging, the specimen is magnified primarily through the objective lens. Each objective lens has an associated depth of field, sometimes also referred to as the depth of focus. Nikon defines depth of field as the distance from the nearest object plane in focus to that of the farthest plane also simultaneously in focus. In microscopy depth of field is very short ranging from approximately 0.2 to 55 micrometers (μm). It is appreciated that, when imaging a transparent specimen, any object within the depth of field will be imaged. For example, using the data in Ref.6, a 4× objective has a depth of field of 55.5 μm, and, therefore, when it is focused on a transparent specimen with the nearest in-focus object plane at the surface of the specimen, all objects within the top 55.5 μm of the specimen will be imaged. This is not desirable when, for example, the area of interest is only the top 5 μm of the specimen. Therefore, there is a need in the art for a microscope system that allows a microscope to be automatically focused on a transparent or translucent specimen at a desired depth, negating, as desired, the remainder of the depth of field of the objective lens. There is a need in the art to provide microscope systems and methods that allow for automatically imaging a transparent or low contrast specimen at a desired depth.